One major aim of this research project is to determine how the conformation of a circular DNA molecule affects its template activity in the Escherichia coli RNA polymerase catalyzed reaction. Specifically we intend to determine whether the same sites of initiation are used on the active form of PM-2 DNA containing 50 negative supercoils as are used on an inactive form containing no supercoils. Analyses will be made of initial nucleotide sequences of the RNAs and of the identity of regions on DNA to which these RNAs are complementary. In addition, we intend to determine the degree to which RNA synthesis is selective in non-homologous transcription systems. RNA synthesized from PM-2 DNA with Pseudomonas BAL 31 RNA polymerase will be compared with that synthesized with E. coli RNA polymerase. A similar complementary study will be made using coliphage DNAs. We will also investigate the effects of solvent changes on template activity of circular DNAs and determine how the activity of the isolated folded chromosome of E. coli compares with the activity of highly purified E. coli DNA. A second major aim is to determine the mechanism of action of the E. coli rho termination factor. We will make a systematic enzymological analysis of the rho associated nucleoside triphosphatase activity in order to determine its relationship to the rho termination function. This alternative assay will also be used to estimate the amounts and location of rho factor inside of cells and to devise an alternative purification procedure for the factor. Finally, we also intend to investigate the importance of rho in suppression transcription initiated by false starts.